Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon
Nicoletta Tekki-Kessaris1,
Rachel Woodruff1,
Anita C. Hall1,*,
William Gaffield5,
Shioko Kimura4,
Charles D. Stiles3,
David H. Rowitch2 and
William D. Richardson1,
1 Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6AE, UK
2 Department of Pediatric Oncology and
3 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
4 Laboratory of Metabolism, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA
5 Western Regional Research Center, ARS, United States Department of Agriculture, Albany, CA 94710, USA
* Present address: Laboratory of Molecular Neurobiology, Karolinska Institute, Berzeliusväg 3, 17177 Stockholm, Sweden
View larger version (69K):
[in a new window]
|
Fig. 1. Expression of Pdgfra, Shh and Ptc1 in the embryonic rat forebrain. (A-C), E13.5 anterior forebrain, coronal sections. Pdgfra is expressed in the neuroepithelium and adjacent SVZ at the boundary between anterior hypothalamus and MGE (arrowhead in A) within broader domains of Shh (B) and Ptc1 (C) expression. There is an additional domain of Shh and Ptc1 expression in the preoptic recess (arrow in B), but there is no Pdgfra expression in this region. (D) Coronal and (E) parasagittal sections of E14.5 anterior forebrain. Pdgfra is widely expressed outside the nervous system (D). In the ventral forebrain, Pdgfra is strongly expressed in the VZ and SVZ of the anterior hypothalamus, extending dorsally into the MGE (D), but is not expressed in the LGE or cortex at this stage. Strong expression is also observed in the primordia of the choroid plexus (arrow in D). In the SVZ, two categories of Pdgfra+ cells are intermingled: closely packed cells that express relatively low levels of Pdgfra and smaller cells that are more intensely labelled (arrowheads in E). (F) Diagram to show the plane of section of A-D and the location of the field shown in E. MGE and LGE, medial and lateral ganglionic eminences; Cx, cerebral cortex; Tel, telencephalon; Di, diencephalon; 3V, third ventricle.
|
|
View larger version (30K):
[in a new window]
|
Fig. 2. Scattered Pdgfra+ cells spread into the cerebral cortex in lateral-to-medial and ventral-to-dorsal directions. (A-C) Coronal sections through the neocortex at different ages, hybrized in situ with a probe for Pdgfra. (A) At E14.5, there are no Pdgfra+ cells in the developing cortex. (B) By E17.5, intensely labelled Pdgfra+ cells are present within the developing cortical plate and presumptive sub-cortical white matter at the lateral margin of the cortex as well as the medial cortex (arrowheads in B). (C) By E20.5, there are numerous Pdgfra+ cells in the cortex, especially in the sub-cortical white matter. At all stages, Pdgfra is also expressed by the meninges and skull.
|
|
View larger version (48K):
[in a new window]
|
Fig. 3. Overlapping expression of Pdgfra, Sox10, Olig1, Olig2 and Shh in the ventral forebrain and neocortex. (A-E) Serial coronal sections of rat E14.5 forebrain. The four presumptive oligodendrocyte lineage markers show overlapping but non-identical patterns of expression in the neuroepithelium, SVZ and mantle zones of the anterior hypothalamus and MGE. Their expression domains also overlap Shh expression in the neuroepithelium (E). The neuroepithelial expression of Olig2 extends further than the rest, through the MGE and LGE (D). (F) Diagram to indicate the approximate position of sections A-E. (G-K) Serial coronal sections through the rat cortex at E18.5. Cells expressing Pdgfra, Sox10, Olig1 and Olig2 have a similar scattered distribution in the cortex at this stage. Shh cannot be detected in the cortex (K). The position of sections G-K is indicated in L. Cx, cortex; Th, thalamus; Hy, hypothalamus; BG, basal ganglia.
|
|
View larger version (76K):
[in a new window]
|
Fig. 4. Pdgfra+ cells from embryonic rat brain differentiate in vitro into GC+ oligodendrocytes or (GFAP+, A2B5+) type-2 astrocytes. These cells were immunoselected from whole brain, but similar results were obtained with cells from cortex or basal ganglia. (A) More than 99% of the process-bearing cells were NG2+. These cells were also (PDGFRA+, A2B5+, O4-, GC-) (not shown). (B) After culturing for a further 2-3 days in the presence of 0.5% FCS without added PDGF-AA more than 99% of the cells differentiated into GC+ oligodendrocytes. (C,D) If the immunoselected cells were instead cultured in the presence of 10% FCS, more than 99% of the cells became GFAP+ astrocytes (C), many of which also labelled with A2B5 (D). Scale bar, 50 µm.
|
|
View larger version (42K):
[in a new window]
|
Fig. 5. Hedgehog signalling is required for oligodendrogenesis in the basal forebrain. Dissociated cells of mouse E10.5 ventral forebrain were cultured in defined medium for up to 8DIV in the presence or absence of the HH inhibitor cyclopamine. The cultures were immunolabelled with polyclonal anti-NG2. (A) Numerous NG2+ cells developed after 8DIV in the control. (B) Their development was strongly inhibited by cyclopamine. (C) The experiment was quantified by counting NG2+ cells in more than ten randomly selected fields (x63 microscope objective). Four independent experiments gave similar results, one of which is illustrated (mean ± s.d.).
|
|
View larger version (109K):
[in a new window]
|
Fig. 6. Pdgfra+ OLPs fail to develop in the anterior hypothalamic neuroepithelium of Nkx2.1 null mice. (A,B) Adjacent coronal sections from the telencephalon of wild-type E12.5 embryos. Pdgfra+ OLPs arise in the ventral forebrain (arrow in A) near the region of Shh expression (arrowhead in B). Shh is also expressed in the diencephalon in the zona limitans intrathalamica (arrow in B) but no Pdgfra+ cells are visible there. (C, D) Adjacent coronal sections from the telencephalon of E12.5 Nkx2.1 null embryos showing a complete absence of Pdgfra+ OLPs (arrow in C) and Shh expression (arrowhead in D) in the ventral forebrain. In contrast, expression of Shh in the zona limitans intrathalamica is maintained in the Nkx2.1 null (arrow in D).
|
|
View larger version (38K):
[in a new window]
|
Fig. 7. Hedgehog-dependent development of OLPs in ventral forebrain cultures of wild-type and Nkx2.1-/- mice. Dissociated ventral forebrain cells from E13.5 wild-type and Nkx2.1-/- mice were cultured in defined medium for up to 8DIV in the presence or absence of the HH inhibitor cyclopamine (cyc, 1 µM). The cultures were immunolabelled with polyclonal anti-NG2 to visualize OLPs and the average number of NG2+ cells per 20 fields (x63 microscope objective) was determined for different times in culture. (A) Wild-type cultures contained NG2+ process-bearing cells from the outset, whereas Nkx2.1 null cultures did not develop OLPs until 4-6DIV. The development of OLPs in Nkx2.1-/- cultures was strongly inhibited by cyclopamine. Asterisks denote that no NG2+ progenitors were detected. (B) Transcripts for SHH and IHH but not DHH could be detected by RT-PCR throughout the culture period. Molecular size markers (M) are 200 bp and 400 bp. The sizes of PCR products were as predicted: Shh, 242 bp; Ihh, 267 bp; Dhh, 311 bp; Gapdh, 382 bp.
|
|
View larger version (119K):
[in a new window]
|
Fig. 8. Rat cortical precursors have latent oligodendrogenic capacity in vitro. Fragments of E15.5 rat cortex or MGE were cultured as explants in collagen gels in basal defined medium for up to 6DIV. The explants were then fixed and immunolabelled with anti-NG2 antibody. (A,B) Cortical explants did not develop NG2+ progenitors until 6DIV. (C,D) In contrast, after 4DIV explants of MGE (C) contained process-bearing NG2+ cells and these increased in number and complexity by 6DIV (D).
|
|
View larger version (20K):
[in a new window]
|
Fig. 9. Hedgehog-dependent development of OLPs in neocortical cultures. (A) Dissociated cells from E15.5 wild-type rat neocortex were cultured in defined medium for up to 8DIV in the presence or absence of cyclopamine (cyc, 1 µM). The cultures were immunolabelled with polyclonal anti-NG2 to visualize OLPs and the number of NG2+ cells per 18 randomly chosen fields (x63 microscope objective) was determined after different times in culture. OLPs did not develop in E15.5 rat cortical cultures until 4-6DIV (see Fig. 8), but after that the number of OLPs increased, peaking after 7DIV. (B) Production of OLPs was inhibited strongly by cyclopamine (A) in a dose-dependent manner. (C) Transcripts encoding SHH and IHH could be detected by RT-PCR as soon as 1 hour after cell dissociation (lane 0), and throughout the culture period up to 8DIV. mRNA coding for DHH was also detected weakly after dissociation and after 8DIV but not at intermediate times. Sizes of PCR products were as predicted (see Fig. 7).
|
|
© The Company of Biologists Ltd 2001
